Emerging avian influenza viruses pose an increasing threat to domestic poultry and human health. The influenza virus hemagglutinin (HA) is an attractive drug target because they are essential for viral entry, and indispensable for virus replication. We hypothesize that fusion inhibitors currently under development in this program will be potent against diverse virus strains, especially HPAI viruses. Specific aims to further develop these inhibitors are:Aim 1. Identify fusion inhibitors that are potent against diverse strains of H5N1 influenza viruses. Our preliminary studies have identified a group of lead compounds that have EC50 values in single digit nanomoles across diverse influenza virus strains including H5N3 (vaccine strain), H3N2, H1N1 and type B. To identify potential candidates for preclinical and eventual clinical studies against HPAI H5N1 viruses, we propose to develop a library of analogs based on the initial lead. We propose to measure the inhibitory potencies of candidate compounds against diverse strains of H5N1 influenza viruses. Lead compounds identified from these studies will be advanced to preclinical studies as a component of Program 10. Aim 2. Determine the mechanisms of action of HA protein inhibitors.Our preliminary data indicate that the fusion nhibitors we have developed alter the structure of HA. For compounds identified in Aim 1 that are potent against H5N1 viruses, we will determine which step of virus entry is blocked using biological and biochemical assays. To map the binding sites of compounds, we will co-crystallize the HA protein with bound inhibitor, and we will also generate and characterize resistant variants. Recombinant HA will be treated with different proteases in order to map the conformationally sensitive regions. Aim 3. Determine if drug combinations that nclude HA inhibitors provide advantages over existing single-agent therapies in protecting against disease and avoiding drug resistance. A widespread use of HA inhibitors could result in the emergence of drugesistant viruses, similar to the acquisition of drug resistance that has been observed for neuraminidase (NA) and M2 inhibitors. We hypothesize that the use of HA inhibitors in combination with existing NA and/or M2 nhibitors will help counteract drug resistance and decrease the severity of infectious disease.